U.S. patent application number 10/512972 was filed with the patent office on 2005-10-20 for device for the amplification of dna, comprising a microwave energy source.
Invention is credited to Corbett, John Michael, Corbett Jr, John Michael.
Application Number | 20050233324 10/512972 |
Document ID | / |
Family ID | 3835631 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233324 |
Kind Code |
A1 |
Corbett, John Michael ; et
al. |
October 20, 2005 |
Device for the amplification of dna, comprising a microwave energy
source
Abstract
The invention relates to a device for the amplification of DNA
in a reaction mixture, the device (1) comprising a heated chamber
(2) including a rotor (3) for holding a plurality of reaction
vessels for reaction mixtures, a drive means for the rotor, a
microwave energy source (8) with means for controlled delivery of
said energy to the reaction mixtures, and a system (14-16) for
determining denaturation of double-stranded DNA. The invention also
provides a method for the amplification of a nucleic acid strand.
In the first step of the method, a reaction mixture is formed
comprising the target nucleic acid strand, nucleotides, a primer, a
thermostable nucleic acid polymerase, and, if necessary, a reagent
for the detection of denaturation of double-stranded DNA. In the
second step, the mixture is incubated at a temperature which allows
synthesis of a nucleic acid strand complementary to the target
nucleic acid strand. The third step comprises denaturing
double-stranded DNA formed in the second step by microwave
energisation of the reaction mixture with monitoring of the mixture
to determine the denaturation end point. The reaction mixture is
allowed to cool to a temperature at which primer anneals to the
target nucleic acid strand in the fourth step. The second to fourth
steps are repeated until a desired level of amplification is
achieved.
Inventors: |
Corbett, John Michael;
(Sanctuary Cove, AU) ; Corbett Jr, John Michael;
(Bellevue Hill, AU) |
Correspondence
Address: |
STEPTOE & JOHNSON LLP
1330 CONNECTICUT AVENUE, N.W.
WASHINGTON
DC
20036
US
|
Family ID: |
3835631 |
Appl. No.: |
10/512972 |
Filed: |
June 13, 2005 |
PCT Filed: |
May 1, 2003 |
PCT NO: |
PCT/AU03/00515 |
Current U.S.
Class: |
435/6.11 ;
219/679; 435/287.2; 435/91.2 |
Current CPC
Class: |
C12Q 2523/313 20130101;
C12Q 2523/313 20130101; C12Q 2563/173 20130101; B01L 7/52 20130101;
B04B 15/02 20130101; B01L 2300/0803 20130101; B01L 7/5255 20130101;
C12Q 1/686 20130101; C12Q 1/6816 20130101; G01N 2035/0444 20130101;
B01L 2300/0627 20130101; C12Q 1/686 20130101; C12Q 1/6816 20130101;
B01L 2300/1844 20130101; B01L 2200/147 20130101; B01L 2300/1866
20130101; G01N 2035/00405 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 435/287.2; 219/679 |
International
Class: |
C12Q 001/68; C12P
019/34; C12M 001/34; H05B 006/64 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 2002 |
AU |
PS 2058 |
Claims
1. A device for the amplification of DNA in a reaction mixture, the
device comprising: a heated chamber including a rotor for holding a
plurality of reaction vessels for reaction mixtures; a drive means
for said rotor; a microwave energy source with means for controlled
delivery of said energy to said reaction mixtures; and a system for
determining denaturation of double-stranded DNA.
2. The device of claim 1, wherein said chamber has a lid or
sealable opening for loading the device rotor.
3. The device of claim 1, wherein said heating of said chamber is
by an internal heater or by supplying heated air through at least
one port in a wall of said chamber.
4. The device of claim 1, which further includes temperature sensor
within the chamber which is linked to an associated computer
responsible for controlling the operation of the device.
5. The device of claim 1, wherein said rotor is fabricated from a
plastics material.
6. The device of claim 1, wherein said rotor drive means is a
direct-coupled AC motor, DC motor or stepper motor.
7. The device of claim 1, wherein said microwave energy source
comprises a magnetron that is external to the device chamber with
energy delivered to reaction vessels via a wave guide.
8. The device of claim 1, wherein said system for determining
denaturation of double-stranded DNA is a fluorescence detection
system or an infrared measurement system.
9. The device of claim 1, which further includes a mechanism for
cooling the device chamber.
10. A method for the amplification of a nucleic acid strand, the
method comprising the steps of: i) forming a reaction mixture
comprising said target nucleic acid strand, nucleotides, a primer,
a thermostable nucleic acid polymerase, and, if necessary, a
reagent for the detection of denaturation of double-stranded DNA;
ii) incubating said mixture at a temperature which allows synthesis
of a nucleic acid strand complementary to said target nucleic acid
strand; iii) denaturing double-stranded DNA formed in step (ii) by
microwave energisation of said reaction mixture with monitoring of
said mixture to determine the denaturation end point; iv) allowing
said reaction mixture to cool to a temperature at which primer
anneals to said target nucleic acid strand; and v) repeating steps
(ii) to (iv) until a desired level of amplification is
achieved.
11. The method of claim 10, wherein said method is carried out in a
device according to claim 1.
12. The method of claim 10, wherein said reagent for the detection
of the denaturation of double stranded DNA is a fluorophore.
Description
TECHNICAL FIELD
[0001] The invention the subject of this application relates to
procedures that require the rapid cycling of a reaction mixture
between different conditions. More particularly, the invention
relates to a device and method for use in such cycling procedures.
The device and method are particularly suited for amplifying DNA in
processes involving successive cycles of annealing, polymerisation
and denaturation steps.
BACKGROUND ART
[0002] In a number of applications such as gene analysis and DNA
profiling, it is desirable to multiply the amount of particular
nucleic acid sequences present in a sample. For example, a duplex
DNA segment of up to approximately six thousand base pairs in
length may be amplified many million fold by means of the
polymerase chain reaction (PCR), starting from as little as a
single copy. In this technique, a denatured duplex DNA sample is
incubated with a molar excess of two oligonucleotide primers, one
being complementary to a short strand of the DNA duplex and the
other being identical to a second short sequence upstream of it
(i.e., more 5').
[0003] Each primer anneals to its complementary sequence and primes
the template-dependent synthesis by DNA polymerase of a
complementary strand which extends beyond the site of annealing of
the other primer through the incorporation of deoxynucleotide
triphosphates. Each cycle of denaturation, annealing and synthesis
affords an approximate doubling of the amount of target sequence,
where the target sequence is defined as the DNA sequence subtended
by and including the primers. A cycle is controlled by varying the
temperature to permit successive denaturation of complementary
strands of duplex DNA, annealing of the primers to their
complementary sequences, and primed synthesis of new complementary
sequences. The use of a thermostable DNA polymerase obviates the
necessity of adding new enzyme for each cycle, thus allowing
automation of the DNA amplification process by thermal cycling.
Twenty amplification cycles increases the amount of target sequence
by approximately one million-fold.
[0004] More detailed information regarding the polymerase chain
reaction can be found in standard texts such as PCR Protocols--A
Guide to Methods and Application (M. A. Innis, D. H. Gelfard, J. J.
Sainskey and T. J. White ed's, Academic Press, Inc., San Diego,
1990), the entire content of which is incorporated herein by cross
reference.
[0005] It has been found that the technique of DNA polymerization
requires rapid controlled heating and cooling cycles. The art is
replete with incubators and other devices to achieve this end--see,
for example, International Patent Application No. PCT/AU90/00560
(Publication No. WO 91/07504) and International Patent Application
No. PCT/AU98/00277 (Publication No. WO 98/49340).
[0006] Typically, a device used for PCR consists of a heat
conductive material provided with channels or cavities adapted to
receive vessels in which the reaction is to take place, for example
Eppendorf.TM. tubes. The heat conductive material is then provided
with heating/cooling means.
[0007] Wittwer et al. in Biotechniques 10, 76-82 (1991) state that
in commercial units for automated DNA amplification, temperature
transition rates are usually no less than 3.degree. C. per second
when metal blocks or water are used for thermal equilibration and
samples are contained in plastic micro-centrifuge tubes. A
significant fraction of cycle time is spent heating and cooling the
sample, as opposed to being spent at optimal denaturation,
annealing and elongation temperatures. Extended amplification times
of two to four hours are common and long transition times make it
difficult to determine optimal temperatures and times for each
stage. Instantaneous temperature changes are not possible because
of sample, container and cycler heat capacities.
[0008] In the international application referred to
above--PCT/AU98/00277, the entire content of which is incorporated
herein by cross reference--there is disclosure of a method and
device for thermal cycling of reaction mixtures which at least
partially overcome the temperature transition rate problem referred
to in the previous paragraph. In the PCT/AU98/00277 device and
method, reaction mixture vessels are held in a rotor which rotates
in a controlled temperature environment. Temperature cycling is
effected by heating and cooling of the environment. This allows
more efficient heating and cooling of the reaction mixtures since
transition losses are minimised. Also, the fact that the samples
are spinning ensures that all samples are heated and cooled at the
same rate. Consequently, no equilibration times are required when a
set point temperature is reached.
[0009] While the PCT/AU98/00277 device and method reduces cycle
time, the time is nevertheless too long for use in a linear
amplification process. Such a process is desirable as it allows
amplification of a single DNA or cDNA strand. Other available
amplification devices and methods similarly have cycle times of
such a period that use for linear amplification is not
feasible.
[0010] It would therefore be desirable to have available a device
and method suitable for linear amplification of a nucleic acid
strand.
SUMMARY OF THE INVENTION
[0011] An object of the invention is to provide a device and method
for the cycling of a reaction mixture in which the cycle time is
such that it allows the device and method to be used for the linear
amplification of DNA.
[0012] According to a first embodiment of the invention, there is
provided a device for the amplification of DNA in a reaction
mixture, the device comprising:
[0013] a heated chamber including a rotor for holding a plurality
of reaction vessels for reaction mixtures;
[0014] a drive means for said rotor;
[0015] a microwave energy source with means for controlled delivery
of said energy to said reaction mixtures; and
[0016] a system for determining denaturation of double-stranded
DNA.
[0017] According to a second embodiment of the invention, there is
provided a method for the amplification of a nucleic acid strand,
the method comprising the steps of:
[0018] i) forming a reaction mixture comprising said target nucleic
acid strand, nucleotides, a primer, a thermostable nucleic acid
polymerase, and, if necessary, a reagent for the detection of
denaturation of double-stranded DNA;
[0019] ii) incubating said mixture at a temperature which allows
synthesis of a nucleic acid strand complementary to said target
nucleic acid strand;
[0020] iii) denaturing double-stranded DNA formed in step (ii) by
microwave energisation of said reaction mixture with monitoring of
said mixture to determine the denatur-ation end point;
[0021] iv) allowing said reaction mixture to cool to a temperature
at which primer anneals to said target nucleic acid strand; and
[0022] v) repeating steps (ii) to (iv) until a desired level of
amplification is achieved.
[0023] It will be appreciated from the foregoing definitions of
embodiments of the invention that the invention relies on microwave
energy to denature DNA rather than heat as in conventional
procedures. In this way, faster cycles are possible as there is no
need to externally heat reaction mixtures to denature DNA or to
delay the primer annealing and polymerisation steps while the
mixture cools to the set temperature for primer annealing and
polymerisation.
[0024] With regard to the first embodiment of the invention, the
device is essentially the same as that described in PCT/AU98/00277,
but modified to include the microwave energy denaturisation
feature. In broad terms, the device chamber can be any suitable,
typically insulated, container for the internal device components
and for association of ancillary components therewith. The chamber
advantageously has a lid or sealable opening for loading the device
rotor.
[0025] Heating of the chamber can be by any of the means used for
controlled heating of automated reaction devices. Typically,
heating is by a heater located within the chamber with circulation
of heated air within the chamber aided by a fan. Alternatively,
heated air can be supplied to the chamber from a port or ports in a
chamber wall.
[0026] A device according to the invention will normally have a
temperature sensor within the chamber which is linked to an
associated computer responsible for controlling the operation of
the device. Through sensing of the chamber temperature, heater
operation can be controlled via the computer.
[0027] With regard to the device rotor, this can be any suitable
rotor provided that it is not fabricated from a metals material.
Rotors are also preferably not heat conductive or electrically
conductive. This is to avoid heating of the rotor during microwave
energisation of reaction mixtures. Heat accumulation by the rotor
would prevent maintenance of the reaction mixture at the annealing
and polymerisation temperature. Advantageously therefore, rotors
are fabricated from a plastics material. Rotors are typically a
flat disc with an annular ring forming an outward portion thereof
which is angled upwardly and has apertures therein for holding a
plurality of reaction tubes. The rotor can be a disposable item
which is used for a single set of amplifications.
[0028] The rotor drive means can be any drive means used for rotor
devices in scientific equipment. For example, the drive means can
be a direct-coupled AC motor, a DC motor, or an AC motor that
drives the rotor via a gearbox or pulleys or the like. Preferably,
the drive means is a direct-coupled AC motor, DC motor or stepper
motor with the motor external to the chamber.
[0029] The microwave energy source typically comprises a magnetron
that is external to the device chamber with energy delivered to
reaction vessels via a wave guide or any other means for delivering
microwave energy known to those of skill in the art.
[0030] The system for determining denaturation of double-stranded
DNA can be any system known to those of skill in the art.
Typically, the system is a fluorescence detection system which will
be described in greater detail below. However, the system can also
by an infrared measurement system comprising a standard
commercially-available IR detection element mounted on the side of
the device chamber and focused on the tips of the reaction vessels
to monitor vessel temperature. If necessary, compensation can be
made for differences between the temperature of a reaction mixture
at the point of DNA denaturation and the temperature of the vessel
wall at that point.
[0031] The light source and detector for the fluorescence detection
system comprises standard components known to those of skill in the
art. For example, the light source can be an LED, a laser light
source or a halogen lamp, with an appropriate filter to provide
light of an appropriate wavelength for excitation of the
fluorophore in the reaction mixture.
[0032] Emitted fluorescence is typically filtered and then measured
by a photomultiplier tube, CCD array, photodiode or CCD camera. The
light source and detector are advantageously linked to the
associated computer whereby the application of microwave energy is
controlled. It will be appreciated by one of skill in the art that
on denaturation of double stranded DNA, there will be a drop in the
fluorescence of the reaction mixture at which point application of
microwave energy can be terminated. Advantageously, a
double-stranded DNA reference standard--for example, genomic DNA or
cDNA--of a known concentration can be included in reaction mixtures
for the purposes of monitoring when denaturation occurs.
[0033] Devices according to the invention can optionally include a
mechanism for cooling the device chamber. Such a mechanism
typically comprises an air supply to the chamber wherein the air is
either at ambient temperature or less than ambient by passage
through or over a cooling means.
[0034] The fluorescence detector used to monitor denaturation of
double stranded DNA can also be used to monitor the progress of a
reaction. For example, the level of fluorescence prior to
denaturation can be used to assess the amount of DNA synthesised.
Devices can further-more include additional monitoring equipment
such as a spectrophotometer or photometer. The additional
monitoring equipment can be dedicated to assessing the progress of
a reaction while the fluorescence detection system acts solely as a
denaturation monitor.
[0035] As alluded to above, operation of the device at the
beginning and end of the amplification, and through each
denaturation, annealing and polymerisation cycle is advantageously
controlled by an associated computer. The computer can control such
operations as:
[0036] rotor start up and speed (any speed greater than 100 rpm is
suitable but typically rotors are rotated ate 500 rpm);
[0037] the annealing and polymerisation temperatures and the time
for each of these steps; the period of application of microwave
energy during the denaturation step;
[0038] processing of data generated by the denaturation system and
any system used to measure the amount of DNA synthesised;
[0039] rotor braking; and
[0040] cooling of the device chamber if necessary such as at the
beginning and end of the amplification.
[0041] It will be appreciated that the computer can be used to
control any other equipment or mechanisms associated with the
device.
[0042] With regard to the second embodiment of the invention, the
method is advantageously carried out using the device according to
the first embodiment. It will be appreciated however that it is not
essential that the device according to the invention be used for
the method which is amenable to adaptation to other devices.
[0043] With regard to step (i) of the method, the reaction mixture
can be any mixture used for an amplification reaction. Typical
reaction mixtures are described, for example in standard reference
texts such as PCR: a Practical Approach (M J McPherson et al.,
Ed's), IRL Press, Oxford, England, 1991, and numerous brochures
provided by suppliers of amplification reagents and
consumables.
[0044] The term "target nucleic acid strand" is used herein in the
context of a linear amplification to denote one of the strands of a
double stranded DNA molecule (genomic or cDNA) to which the primer
will anneal to provide a complementary sequence thereto in the
amplification reaction. However, the method can be used for non
linear amplification of double stranded DNA in which case the
reaction mixture will include a primer for each strand of the DNA
molecule. Exponential amplification of the double stranded target
will result with repetition of the steps of the method.
[0045] With regard to the reagent for the detection of the
denaturation of double stranded DNA which can be included in
reaction mixtures, this will be a fluorophore required for the
optical temperature calibration or fluorescence detection systems
referred to above. The intercalating fluorophore can be any of
those known to persons skilled in the art and include ethidium
bromide and SYBR.TM. Green.
[0046] The annealing and polymerisation steps can be carried out at
any of the temperatures and for the times used in known
amplification procedures.
[0047] In current instruments for performing PCR and other linear
and non-linear DNA amplification reactions, energy is supplied to
the reaction mixture via the reaction vessel wall (glass or
plastic) which acts as an insulator. This process usually takes 1
to 2 minutes for a single cycle (55.degree. C.-95.degree.
C.-55.degree. C.). In a typical reaction, 30 to 50 cycles are
required.
[0048] The device according to the invention allows a reaction
vessel to be held with its wall at typically 50-65.degree. C. with
energy for the denaturation being supplied to the reaction mixture
by energizing the magnetron (typically for several seconds). As the
reaction vessel wall is held at the typical annealing temperature
of 50-65.degree. C., the mixture returns to that temperature
shortly after application of microwave energy is terminated due to
the fact that the reaction vessel does not absorb an appreciable
amount of microwave energy. This reduces the cycle time to 6 to 10
seconds.
[0049] Because of the fast cycle time possible with the device and
method of the invention, a PCR amplification of double stranded DNA
can be performed in minutes rather than hours. More importantly,
linear reactions for the amplification of a single strand of DNA
can be executed in 1 to 2 hours. This is not achievable with
existing devices and methods which require 100 to 1,000 cycles
taking up to 30 hours. The device and method of the invention thus
allow real time assays to be done at least an order of magnitude
faster than currently-available assays.
[0050] Having broadly described the invention, a device will now be
exemplified with reference to the accompanying drawing briefly
described hereafter.
BRIEF DESCRIPTION OF THE DRAWING
[0051] FIG. 1 is a schematic cross section of a device according to
the invention.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0052] With reference to FIG. 1, device 1 comprises a chamber 2
having rotor 3 which is driven by a stepper motor not shown in the
drawing. Chamber 2 also includes a radial heater 4 and a fan 5 for
distributing heated air throughout the chamber. Heater 4 and fan 5
are mounted to hinged lid 6 of the device, which lid can be pivoted
out of the way to gain access to rotor 3. Rotor 3 has a plurality
of holes for holding reaction vessels, one of which vessels is item
7.
[0053] Device 1 also includes a magnetron 8 from which microwaves
can be directed via wave-guide 9 to reaction vessels such as 10 as
they pass the microwave emission point 11. A light source 12 is
provided for illuminating a reaction vessel as it passes through
beam 13. Light 14 emitted from reaction vessel 7 passes through
filter 15 to be detected by photomultiplier tube 16.
[0054] Device components such as the rotor drive, heater 4, fan 5,
magnetron 8, and light source 12, are controlled by a computer not
shown in the drawing.
[0055] Operation of the device is as follows. Reaction mixtures are
dispensed into reaction vessels using manual pipettors or automated
robotic pippetting means and heated to the annealing temperature
via heater 4 and fan 5 under the control of the associated
computer. Rotor 3 is rotated at greater than 100 rpm under the
control of the computer during this step and subsequent steps to
average reaction vessel temperatures.
[0056] On command from the computer to denature double stranded DNA
formed in the reaction mixture, magnetron 8 is activated and
microwave energy transferred to reaction mixtures via wave guide 9.
At least one reaction mixture contains an intercalating dye such as
ethidium bromide or SYBR.TM. Green. The dye is excited by light
source 12 and fluorescence measured by photomultiplier tube 16
after selection of light of the appropriate wavelengths by filter
15.
[0057] With denaturation of the double stranded DNA, fluorescence
emission diminishes and on reaching a present level causes the
computer to deactivate magnetron 8.
[0058] On termination of application of microwave energy, the
reaction mixture quickly returns to the annealing temperature
maintained within chamber 2 by heater 3 and fan 4. The cooling of
the annealing temperature takes only seconds as the reaction vessel
per se is not heated by the microwave energy--only the reaction
mixture is acted on by that energy.
[0059] At the annealing temperature, the progress of the reaction
can be monitored by way of a fluorescent probe present in reaction
mixtures or by measuring the increased energy of the intercalating
referred to above dye. This monitoring is by way of light source
12, filter 15 and photomultiplier tube 16. Results can be recorded
by the computer.
[0060] It will be appreciated by a person of skill in the art that
many changes can be made to the device and its use as exemplified
above without departing from the broad ambit and scope of the
invention.
[0061] The term "comprise" and variants of the term such as
"comprises" or "comprising" are used herein to denote the inclusion
of a stated integer or stated integers but not to exclude any other
integer or any other integers, unless in the context or usage an
exclusive interpretation of the term is required.
[0062] The reference to the publications cited in the Background
Art section of this specification is not an admission that the
disclosures constitute common general knowledge in Australia.
* * * * *